The mechanism of somatic hypermutation in the variable region of immunoglobulin genes expressed in mammalian B ceUs is a major unexplained phenomenon in the generation of diversity in the immune system. To evaluate possible mechanisms, the distribution of somatic mutations was examined for a group of five cloned, rearranged, somatically mutated VH genes generated in C57BL/6j mice. The DNA sequences both within and flanking rearranged antibody variable-region genes (VLJL and VHDJH) expressed in B lymphocytes can mutate at a very high rate (14; for recent reviews, see reference 28). This type of genetic variation is termed somatic hypermutation to distinguish it from the other more familiar processes of immunoglobulin diversification, such as junctional diversity, combirnatorial recombination, and the association of fully assembled heavy and light polypeptide chains.The mechanism of hypermutation in mammalian B cells is unknown, but several models have been proposed. These can be segregated into those models in which the DNA is the direct substrate for mutation (5,8,14,17), including gene conversion (26), or those in which the DNA becomes hypermutable as a consequence of gene expression (i.e., transcription-based models [41]). In the first type of model, the DNA is mutated directly either via specific nicking and error-prone repair enzymes (8, 14), through errors introduced by misalignment of replicating DNA templates directed by inverted or direct repeat sequences (17), or via localized amplification of DNA replication which generates errors at the normal rate but in greater number in a localized region (5). Alternatively, the model invoking gene conversion envisages that sequence information donated by other related genes is incorporated by recombination into the rearranged V gene (26). However, these DNA-based models do not state how the enzymes display the specificity which limits the mutational process to the DNA within and around rearranged V genes (41). Known DNA polymerases also display a very high copying fidelity in vivo (24,31,32). Thus, the DNA-based models also require the presence of new enzymes with V-region specificity.A second type of model based on gene expression ac- (15,18,20,22,30,39). They are not found in constant region sequences (15) and are found rarely in unrearranged germ line V genes (44). It is important now to precisely define the distribution of somatic mutations in and around rearranged V genes, particularly in the 5' upstream region, because this will help to identify the target region of the DNA and therefore influence ideas on the type of mutational mechanism involved. In this work, we contribute to a better definition of this target region by comparing DNA sequences of related sets of hypermutated VH genes with their germ line counterparts. MATERIALS AND METHODSCloning and sequencing rearranged somatically mutated VH genes. Genes derived from the VH186.2 germ line gene 5187 on May 12, 2018 by guest

The aim of this study has been to determine the distribution of somatic mutations in the 5' flanking regions of rearranged immunoglobulin heavy chain variable region genes (VDJ). We sequenced the 5' flanking region in 12 secondary immune response antibodies produced in C57BL/6j mice against the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken-gamma-globulin. In these and previously published sequences, almost 97% of the mutations occurred in the transcribed region of the gene, and only a minority of genes (5/29) contained mutations upstream of the transcription start (cap) site. No potential germ-line donor was found for a cluster of five base changes previously found in a single heavy chain gene, 3B62. However, the uniqueness of this mutational cluster and its distance from the normally mutated region suggests that the nucleotide changes may not be due to the normal mutator mechanism. Thus, as this was the only instance of somatic mutations that far upstream of the promoter/cap site region, the reverse transcriptase model for somatic hypermutation is still a possibility. The data are consistent with a mutational mechanism that requires transcription of the rearranged target V(D)J gene which appears to result in the generation of a positively skewed asymmetrical distribution of somatic mutations. A single mode is centered near the V(D)J and a long tail extends into the 3' non-translated region of the J-C intron. Two classes of model could explain this mutation distribution pattern: those where transcription products (RNA, cDNA) are the direct mutational substrates, or those that postulate local unfolding of the chromatin around a V(D)J rearrangement directly exposing the DNA of the transcribed region to specific mutational enzymes.

The mechanism of somatic hypermutation in the variable region of immunoglobulin genes expressed in mammalian B cells is a major unexplained phenomenon in the generation of diversity in the immune system. To evaluate possible mechanisms, the distribution of somatic mutations was examined for a group of five cloned, rearranged, somatically mutated VH genes generated in C57BL/6j mice. These mutated VH genes were sequenced and compared with their germ line counterparts from a point approximately 550 base pairs upstream of the transcription start site to an EcoRI site some 1,200 base pairs downstream of JH-4. The location of the transcription start (cap) sites was also precisely determined. Most (greater than or equal to 94%) of the 118 mutations scored occurred between the transcription start site and the distal end of JH-4. However, seven mutations occurred upstream of the transcribed region, and at least four were found downstream of JH-4. The target region for the mutator mechanism therefore clearly extends into the 3' nontranslated and 5' nontranscribed regions. Thus, models which propose the transcribed region of the DNA as the sole substrate for the mutation process are not ruled out but are inadequate to explain the upstream distribution of somatic mutations.